Field of the Invention
The invention concerns a method and a device for determining hydrogen peroxide concentrations in fluids, in particular an improved determination of hydrogen peroxide concentrations in blood, sweat, urine, or milk.
The determination of substances, specifically in the presence of other substances—some of which are interfering substances—is important for many areas of application, especially in medical diagnostics. For example, determining glucose in the blood is critically important for the treatment of diabetes. Treatment promises to be especially successful when blood sugar is checked at regular intervals by the patient himself. To do this, the patient pricks his finger with a lancing device to obtain a drop of blood, which he applies to a disposable biosensor. The biosensor is located in a measurement device that performs the measurement and analysis. After 10 to 30 seconds, the display shows the blood sugar reading, which the patient uses for ongoing monitoring and/or precise insulin dosing. This necessitates an accurate blood sugar measurement. Occasional erroneous measurements can lead to acute complications such as coma. Repeated erroneous measurements cause a persistent unphysiologically high blood sugar level, which leads to blindness, amputations, kidney failure and myocardial infarction.
Prior Art
A number of methods are known for determining blood sugar levels. These methods generally fall into two categories: optical methods and electrochemical methods. In the case of optical methods, reflectance spectroscopy or absorption spectroscopy are used to detect the quantity of chromophores formed in the reaction with glucose in the blood. The intensity of the color change is proportional to the blood sugar content.
Electrochemical methods use amperometric or coulometric techniques to determine the blood sugar content. The application possibilities and, above all, the performance of electrochemical methods are limited by the multitude of interfering substances in the blood (urea, amino acids, ascorbic acid, medications, etc.), since these substances can also be converted at the electrodes, thus producing erroneously elevated readings. The same applies to other variables, such as hematocrit, for example, which also can differ from measurement to measurement. The hematocrit (HCT) is the percentage of red blood cells in whole blood (in volume %). Normal hematocrit is between 40 and 45 volume %. In the case of illness or in accident victims with high blood loss, the HCT can be between approximately 22 and 65 volume %. The extent to which this affects measurement of the blood sugar content is described in U.S. Pat. Nos. 5,234,516; 5,288,636; 5,353,351 and 5,385,846, for example.
As a result of the uncertain chemical and physiological composition of the blood or other body fluids, the measurement results from the electrochemical glucose determination using the conventional method show deviations of up to 20% from the actual glucose value.
Disposable electrochemical sensors generally consist of a substrate upon which are formed contacts, lines, and electrodes made of a conductive material. The reaction zones and the contacts to the measurement device are defined by the application of a nonconductive layer. The reaction zone is formed by the electrodes. In general, two electrodes are present, one of them serving as measurement electrode. The other electrode represents the reference electrode and counter electrode. The actual detection reaction takes place at the measurement electrode. For this purpose, an enzyme layer or enzyme membrane is applied either to this electrode or in front of it. The enzyme is used to react specifically with the glucose in the blood. The measurement electrode now measures the concentration of one or more reaction products of the enzyme reaction. The concentration of the reaction products is directly proportional to the substrate concentration as long as the activity of the enzyme is higher than the substrate concentration. In this way, the measurement electrode directly determines the glucose concentration in the blood.
A number of enzyme sensors for measuring glucose in blood or other fluids are described in the literature. Nearly all glucose sensors function according to the following reaction scheme:

The glucose determination can take place either through (1) the consumption of oxygen (O2 electrodes), (2) the hydrogen peroxide formed (H2O2 electrodes), or (3) the rise in pH (pH electrodes). Glucose sensors based on changes in pH have the disadvantage that their measurement behavior is determined by the buffer capacity of the sample.
In the case of O2 and H2O2 electrodes, the measured value is directly proportional to the glucose concentration over a range that is large to a greater or lesser extent. The measurement range is determined by the membrane's permeability to glucose and oxygen. High permeability of the membrane makes for high sensitivity, while low permeability reduces the sensitivity but extends the measurement range. At high glucose concentrations, the measurement range may be limited by insufficient enzyme activity. However, this circumstance can be kept in check through the use of excess enzyme. It is important to keep in mind here that autoinhibition can occur with large enzyme quantities, especially in the case of glucose oxidase.
Without a membrane, the measurement range is limited to a maximum of approximately 200 mg/dl by the transport rate of oxygen to the enzyme. But in diabetes treatment, blood sugar levels between 200 and 500 mg/dl are commonplace. To reach this value without sacrificing sensitivity owing to thick membranes, oxygen-independent glucose sensors were developed. In these sensors, molecules other than oxygen transport the electrons from the enzyme redox center to the electrode surface. These molecules are known as mediators M. The mechanism proceeds in accordance with the following scheme:Glucose+GODox→δ-glucolactone+GODred GODred+2Mox→GODox+2Mred+2H+Mred→Mox+e−(at the electrode)  (II)
Such sensors are oxygen-independent to a certain degree, and even function in anaerobic environments.
In electrochemical glucose determination in a complex medium such as blood or urine, however, the problem arises that numerous other substances contained in the medium can influence the measured current. In the case of direct H2O2 detection at approximately +0.6 V (NHE), a great number of easily oxidized compounds are converted at the same time, which produces false elevated readings. For this reason, the mediators used generally have low redox potentials, which reduces the effect of interfering species on the measurement result. Examples of suitable mediators include benzoquinones and naphtoquinones (EP 0 190 740), substituted flavins, phenazines, phenothiazines, indophenols, substituted 1,4-benzoquinones and indamines (EP 0 330 517), N-oxides, nitroso compounds, hydroxylamines and oxines (EP 0 354 441), soluble ferricyanide/ferric compounds (EP 0 496 730) and phenazinium/phenoxazinium salts (U.S. Pat. No. 3,791,988).
Glucose sensors with mediators have disadvantages, however. All mediators are toxic, for example. The natural reaction of glucose oxidase with oxygen from the medium cannot be prevented, since the affinity of the redox center to O2 is approximately 100 times greater than to Fe(III)CN6, for example. Especially at low glucose concentrations this occurs to a greater degree, and produces measured values that are too low, since the H2O2 formed by the reaction with oxygen is not detected. Moreover, when such sensors are manufactured, it is necessary to ensure that the mediator remains in the oxidized state until the time of measurement. Each reduction, even only a partial reduction, results in an increased background current, since Mred molecules are already present, which of course are also converted. It is known (EP 0 741 186) that mediators tend toward fast reduction, especially at high temperatures and high humidity. Naturally, this decreases the shelf life, and hence the application possibilities for using the sensors as disposable sensors.
Two-electrode systems or three-electrode systems may be used. In both systems, the working electrodes and counter electrodes are typically made of platinum, gold, carbon, or the like. The reference electrode is an Ag—AgCl electrode, a calomel electrode, or made of the same material as the working electrode.
The international patent application with publication number WO 81/03546 discloses a method for measuring the glucose concentration in which a voltage curve (lower limit: −1.0 to −0.6 V; upper limit: +0.7 to +1.1 V) is applied which has dwell times at certain points (especially those where glucose is converted) and at the reversal points. The charge is determined in specific regions. The dwell point and dwell times are chosen such that the charge is proportional to the glucose concentration, independent of interfering substances, primarily urea and amino acids. The regions in which integration is performed are characterized in that glucose always provides only a positive contribution to the total charge, while in contrast the interfering substances make both positive and negative contributions which are thus averaged out of the integration.
However, this method fails when concentrations of interfering substances are high at the same time as glucose concentrations are low.
In sum, then, a method for determining glucose in the blood is desired that works with H2O2 electrodes, needs no mediators, is relatively insensitive to hematocrit and temperature, in which H2O2 detection can take place at a lower voltage range, making it more insensitive to interfering species. At the same time, the sensors should be storable, so that they can be used as disposable sensors.